Method for treating defective dura mater

ABSTRACT

Disclosed are an artificial dura mater and manufacturing method thereof. The artificial dura mater includes electrospun layers prepared by electrostatic spinning, at least one of which is a hydrophobic electrospun layer. Further, above the hydrophobic electrospun layer, there can be at least one hydrophilic electrospun layer. A transition layer can be further included between the hydrophobic and the hydrophilic electrospun layers. Additionally, cytokines and/or medicines can be affixed to either or both of the hydrophobic and the hydrophilic electrospun layers, by way of bio-printing. The disclosed artificial dura mater shows good biocompatibility, enhances dural tissue regeneration, achieves excellent repairing effects, prevents adhesion, allows complete absorption, has good mechanical properties, ensures low infection rates, and can be loaded with therapeutic agents.

TECHNICAL FIELD

The invention is directed to artificial biofilms, in particularartificial dura maters, and to related manufacturing methodology.

BACKGROUND

Dural defect is a common problem during neurosurgery. Opencraniocerebral injuries (industrial, traffic or war-related), tumorinvasion, congenital meninges defects or other cranial diseases can leadto defects of the dura mater. Such defects of the dura mater need berepaired timely so as to prevent leakage of the cerebrospinal fluid,encephalocele, and stress from the barometric pressure. Otherwise, itcan be life endangering.

Currently, although there are many dura substitutes, materials used inthe substitutes can be generally classified into four types: autologousfascia, allograft, natural or synthetic substance, and xenograft.However, clinical application of these materials unavoidably leads toproblems such as high infection rates. According to statistics, theinfection rate for craniotomy is 4%; the dura mater made of pig's smallintestine mucosa gives an infection rate of 3.4%; and the dura matermanufactured by collagen exhibits an infection rate at 3.8%. Because ofthe blood brain barrier, once intracranial infection occurs, theencephalic plasma concentration of anti-infection drugs can hardly reachthe desired level and control of infection becomes difficult. Also, theexisting artificial dura products do not have the capability ofsupplying medicines into the meninges. Therefore, post-operationinfection control is often ineffective.

Moreover, one of the common reasons for dural repair with dura matertransplant is damages to the meninges due to tumor invasion. More thanhalf of brain tumors cannot be completely removed by surgery and thuschemotherapy is necessary after the surgery. Many chemotherapeutic drugsare toxic and cannot pass the blood brain barrier, so that an effectiveconcentration of the drugs cannot be reached resulting in diminishedchemotherapy effects.

Current artificial dural substitute products usually do not containtherapeutic drugs of interest. For instance, due to the densestructures, the autologous fascia can not be loaded with drugsnaturally, and it is difficult to load drugs into allograft orxenograft. However, due to their good malleability, syntheticmaterial-based dural substitutes can be readily loaded with drugs. Onthe other hand, due to the limitation of loading methods of a drug, itis also not easy to load the drug onto artificial dura mater, and yetallow release of the drug upon transplantation to achieve therapeuticobjectives. To date, the common way to load an anti-infection drug ontoan artificial dura mater substitute is to soak the substitute with thedrug. With this method, most of medicine remains on the surface ofartificial dura mater, which is easily lost, making it difficult toachieve controlled release.

It is apparent, therefore, that current artificial dura matersubstitutes have shortcomings. Among these are a high infection rate,poor bio-compatibility, incomplete absorbability, and difficulty inloading medicine and controlling the effective release of the medicine.

SUMMARY OF THE INVENTION

In view of these disadvantages of conventional technology, the presentinvention provides an artificial dura mater that is characterized byexcellent tissue compatibility, ideal anti-adhesion, completeabsorption, good mechanical properties, a low infection rate, and acapability to load a variety of therapeutic substances. An artificialdura mater of the invention is comprised of electrospun layers,including at least one hydrophobic electrospun layer. The inventioncontemplates employing methodology that comprises elctrospinning toprepare such a layer with one or more hydrophobic polymers.

Suitable hydrophobic polymers can be selected for this purpose from thegroup consisting of hydrophobic aliphatic polyester, polyetherester,polyorthoester, polyurethane, polyanhydride, polyphosphazene, andpolyamino acid, and copolymers and mixtures thereof. Thus, thehydrophobic aliphatic polyester can be at least one selected from thegroup consisting of polylactic acid, polyglycolide, polycaprolactone,and polyhydroxybutyrate (PHB). The polyetherester can be at least oneselected from the group consisting of the polydioxanone (PDO),glycol/lactic acid copolymer, and glycol/butylenes terephthalatecopolymer. The polyanhydride is at least one selected from the groupconsisting of poly(sebacic acid-hexadecanedioi acid anhydride), type-Ipolyanhydride, type-II polyanhydride, type-III polyanhydride and type-IVpolyanhydride.

The hydrophobic layer-containing artificial dura mater has a strengthsimilar to that of human dura mater. It can seal and protect a person'sbrain and prevent leakage of the cerebrospinal fluid before a person'sown dura mater is regenerated. The hydrophobic layer does not allowcells' migration or attachment and thus can achieve an anti-adhesioneffect. In practice, more than one hydrophobic layers can be included toprovide different levels of strength

Furthermore, in the artificial dura mater according to the invention, atleast a hydrophilic electrospun layer can be placed on the hydrophobicelectrospun layer. The hydrophilic layer can be prepared byelectrospinning with one or more types of hydrophilic polymers selectedfrom the group consisting of chondroitine sulfate, heparin, agar,glucan, algin, cellulose, modified cellulose, alginate, starch, gelatin,fibrinogen, silk protein, elastin-mimicry peptide polymer, collagen,chitosan, modified chitosan, hydrophilic polyurethane, polyethyleneglycol, polymethylmethacrylate, PHBV, PHBHHx, polyvinyl alcohol, andpolylactide, and mixtures thereof.

When the dura mater is transplanted into the brain, the hydrophobiclayer is placed proximate to the brain surface to take advantage of itsanti-adhesion capability; whereas the hydrophilic layer, which serves asan excellent nanofiber scaffold for the adhesion, migration,proliferation, and differentiation of cells, is placed distant to thebrain. As the hydrophilic layer is prepared with hydrophilic materialsof good bio-compatibility, it can effectively enhance the migration andproliferation of stem cells and fibroblasts, and consequently promotethe growth of autologous dura mater. In practice, the hydrophilic layercan include more than one layer, so as to satisfy different needs.

According to one embodiment of the invention, the artificial dura materalso can have a transition layer between the hydrophobic and hydrophiliclayers. The transition layer is prepared by an electrospinning methodcomprising with one or more polymers, and it has hydrophilicity thatgradually increases from the side proximate to the hydrophobicelectrospun layer to the side proximate to the hydrophilic electrospunlayer. The present of the transition layer can improve the affinitybetween the hydrophobic and hydrophilic layers.

According to another embodiment of the invention, any one or more of thehydrophobic layers, the hydrophilic layers, and the transition layerscan be blended with a cytokine and/or a medicine. Such layers can allowrelease of the cytokine and/or the medicine into local brain tissue upontransplantation, along with the absorption of the polymers. The cytokineand/or the medicine released to the local brain tissue can then beuseful in preventing local infection, adhesion, and/or tumor'srecrudesce, or promoting the restoration of autologous dura mater.

According to yet another embodiment of the invention, the hydrophobiclayer and/or the hydrophilic layer can be affixed with the cytokineand/or the medicine. In this regard, the cytokine can be an agent thatplays a role in adhesion, migration, proliferation, and/ordifferentiation of fibroblast. Thus, it can be selected from the groupconsisting of interleukin, colony stimulating factor, tumor necrosisfactor, platelet derived growth factor, basic fibroblast factor, andcombinations thereof. The cytokine can facilitate the recovery of thedefective dura mater.

A medicine used in accordance with the invention can be one or moreselected from the group consisting of antibiotic, hemostat,anti-adhesion agent, and tumor-resistance drug. These medicines can beplaced onto the artificial dura mater, based on actual needs. Once theartificial dura mater is transplanted, the medicines are releasedgradually to local brain tissues, thereby bypassing the blood brainbarrier, to achieve therapeutic effects during the degradation of thepolymers and regeneration of the dural defect.

Furthermore, the cytokine and/or medicine can be enclosed in a hydrogel.With the adhesion and fixation effects of hydrogel, the medicine can,depending on the local situation, be uniformly or specifically released.The hydrogel can be prepared with one or more selected from the groupconsisting of polysaccharide polymer, polypeptide polymer, and synthetichydrophilic high molecular polymer.

According to one embodiment of the invention, the hydrophobicelectrospun layer of the artificial dura mater is comprised of fibershaving a diameter of 50-1000 nM. According to one embodiment of theinvention, the hydrophobic electrospun layer has pores of a size of lessthan 3 μM, whereas the fibers of the hydrophilic electrospun layer havea diameter of 5-200 μM, and a pore size of 20-200 μM. The pore sizedepends largely on the diameter of the fibers. When the diameter offiber decreases, the pore size reduces. Thus, by controlling the fiber'sdiameter, one can control the pore size of the electrospun layer aswell. The average diameter of a human cell is 10-50 μM. The meningesmainly consist of the fibroblasts and collagen fibers excreted fromfibroblast. Most of the fibroblasts have a diameter of 20-30 μM. Thepore size of the hydrophobic layer is less than 3 μM, which cantherefore prevent the entry of cells and adhesion between dura mater andbrain tissue. The hydrophilic layer has a pore size equal to or largerthan the average diameter of cell, which can promote the entry andmigration of cell.

According to further aspect of the invention, a method for preparing anartificial dura mater is also provided, comprising the following steps:

A) dissolving a hydrophobic polymer into a solvent to obtain ahydrophobic electrospinning solution, wherein the hydrophobic polymer isselected from the group consisting of hydrophobic aliphatic polyester,polyetherester, polyorthoester, polyurethane, polyanhydride,polyphosphazene, polyamino acid and copolymers and the mixtures thereof;

B) producing, by electrospinning, a film-like (or fleece-like)hydrophobic electrospun layer from the hydrophobic electrospinningsolution, thereby preparing the artificial dura mater.

Further, the hydrophobic aliphatic polyester can be at least oneselected from the group consisting of polylactic acid, polyglycolide,polycaprolactone and polyhydroxybutyrate. The polyetherester can be atleast one selected from the group consisting of polydioxanone (PDO),glycol/lactic acid copolymer and glycol/butylenes terephthalatecopolymer. The polyanhydride can be at least one selected from the groupconsisting of poly(sebacic acid-hexadecanedioi acid anhydride), type Ipolyanhydride, type II polyanhydride, type III polyanhydride and type IVpolyanhydride.

The method, applying the principle of electrospinning, forms theartificial dura mater by using a particular polymer. The dura mater caneffectively prevent it from adhering to brain tissue.

To further prevent the adhesion of artificial dura mater to braintissue, the diameter of fibers can be controlled, such that the poresize of the scaffold is also controlled. In this respect, the artificialdura mater inhibits cell migration. The diameter of fibers of thehydrophobic electrospun layer can be controlled within 50-1000 nM.According to one embodiment of the invention, the pore size ofhydrophobic electrospun layer is less than 3 μM.

In the method described above, at step B), the electrospinning ispreformed with a micro-injection pump operated at a velocity of 0.1-5.0milliliters/hour and a high voltage generator operated at a voltage of5-40 kilovolts and with a receiving distance of 5.0-30.0 centimeters.

To achieve a better effect in clinical therapy, the invention alsoprovides a method, alike to electrospinning, to form a hydrophilicelectrospun layer on the hydrophobic layer that comprising the steps of:

a′) Dissolving a hydrophilic polymer into solvent to obtain ahydrophilic electrospinning solution, wherein The hydrophilic polymer isselected from the group consisting of chondroitine sulfate, heparin,agar, glucan, algin, modified cellulose, alginate, starch, cellulose,gelatin, fibrinogen, silk protein, elastin-mimicry peptide polymer,collagen, chitosan, modified chitosan, hydrophilic polyurethane,polyethylene glycol, polymethylmethacrylate, PHBV, PHBHHx, polyvinylalcohol polylactide.

b′) Placing the hydrophilic electrospinning solution, byelectrospinning, on the hydrophobic layer to form the hydrophilicelectrospun layer.

The hydrophilic electrospun layer is placed distant to the brainsurface, in order to promote migration of cells and regeneration ofdural mater. To facilitate the entry of cell, the hydrophilicelectrospun layer has fibers with a diameter of 5-200 μM and pores of asize of 20-200 μM.

In step b′), the formation of hydrophilic electrospun layer, theelectrospinning is preformed with a micro-injection pump operated at avelocity of 0.1-20.0 milliliters/hour and a high voltage generatoroperated at a voltage of 10-40 kilovolts and with a receiving distanceof 5.0-30.0 centimeters.

In the process of electrospinning, generally, the hydrophobic andhydrophilic polymers should be dissolved into appropriate solventsrespectively, to form electrospinning solutions. Often, the solvents arevolatile organic solvents which include but not limited to methanoicacid, acetic acid, ethyl alcohol, acetone, dimethyl formamide, dimethylacetamide, tetrahydrofuran, dimethyl sulfoxide, hexafluoro isopropylalcohol, trifluoroethyl alcohol, dichloromethane, trichloromethane,methyl alcohol, ethyl alcohol, chloroform, dioxane, trifluoroethane,trifluoroacetic acid and mixtures thereof. The volatile organic solventswill quickly volatilize during the process of forming electrospunlayers, and the final electrospun layers will contain no or littleresidual organic solvent which can be removed in the later steps. Insome cases, water can be used as solvent and removed by oven or naturaldryness after the electrospun layers are formed.

Furthermore, the method for preparing an artificial dura mater providedin present invention further comprises, before the hydrophilicelectrospun layer is prepared, forming a transition layer byelectrospinning between the hydrophilic and hydrophobic layers, whereinthe transition layer has hydrophilicity that gradually increase from theside proximate to the hydrophobic electrospun layer to the sideproximate to the hydrophilic electrospun layer. The materials, solventsand electrospinning parameters for preparing the transition layer can bedetermined based on the actual situation and need. The present of thetransition layer can improve the hydrophilicity between hydrophilic andhydrophobic layers.

In one embodiment of the invention, during the formation of eachelectrospun layer by electrostatic spinning, a cytokine and/or amedicine can be added to the corresponding electrospinning solutions.With the blending technique, the blended layers of polymers and acytokine and/or a medicine have a better satisfaction for clinicapplication and a better therapeutic effect.

The method for preparing an artificial dura mater provided in presentinvention further comprises, by bio-printing, forming a distribution ofa cytokine and/or a medicine on the hydrophobic electrospun layer and/orthe hydrophilic electrospun layer. Bio-printing is a technology forprinting a cytokine and/or a medicine onto bio-papers that comprisingscaffolds of the hydrophobic electrospun layers and/or the hydrophilicelectrospun layers.

To make the cytokine and/or medicine distribute evenly and point fixedonto the layers, the cytokine and/or medicine can be enclosed into ahydrogel.

Specifically, the bio-printing comprises the steps of:

a″) admixing a hydrogel solution with cytokine and/or medicine to form asolution;

and

b″) printing the solution onto the hydrophobic electrospun layers and/orhydrophilic electrospun layers using a bio-printing technology.

The hydrogel solution in the invention comprises the aqueous solutionsof polysaccharide polymer, polypeptide polymer synthetic hydrophilicpolymer or mixtures thereof. Wherein the polysaccharide polymer includesbut not limited to starch, cellulose, alginate, hyaluronic acid andchitosan. The polypeptide polymer includes but not limited to collagen,poly-L-lysine and PLGA. The synthetic hydrophilic polymer includes butnot limited topolyacrylic acid, polymethacrylic acid, polyacrylamide andN-isopropyl acrylamide.

The hydrogel is liquid under normal circumstance. At appropriatetemperature or under specific conditions, it can turn to gel for a shorttime by which it has a good adhesion. According to the invention, somehydrogels need a cross-linking agent in participation of reaction.Therefore, the method further comprises, before bio-printing, apretreatment of the hydrophobic and/or hydrophilic layers with asolution comprising a cross-linking agent. With the pretreatment by thesolution comprising a cross-linking, a cross-linking agent is adhere onthe layer or layers. After that, a cytokine and/or a medicine will beadded into a hydrogel solution, and the mixed solution will be placedinto the printer head. While printing, when the hydrogel solution withcytokine and/or medicine reaches the electrospun layers, it solidifiesand adheres to the layers.

In a uniform and stable printing the cytokine and/or medicine can beevenly released. While in a customized printing with varied speeds andlocations for an individual case, the cytokine and/or medicine can bereleased in specific area. The selection of cross-linking agent is basedon the type of hydrogel. For instance, while the hydrogel is sodiumalginate, the cross-linking agent is calcium chloride; while thehydrogel is fibrinogen, the cross-linking agent is thrombin.

Compared with the prior arts, the invention exhibits the followingadvantages:

(1) Its mechanical properties satisfy the requirement for tensilestrength and flexibility, and it is waterproof and anti-adhesion.

(2) The materials comprising of the membranes is free from poison andharmless to human body, have good compatibility, allow completelyabsorption after implantation, which avoids the occurrence of tumor orcancer.

(3) The membrane is not prepared with animal tissues, therefore it canavoid risks such as immune rejection, virus spreading and diseaseinfectiousness.

(4) The designed double layers can prevent adhesion, promote the growthof autologous cells, which, enable an earlier restoration of dura mater.

(5) By incorporating the bio-printing technology, the therapeuticsubstances can be introduced into the membrane and can be released in acontrolled manner after the implantation.

(6) The materials are abundant in source and, cheap in cost andconvenient in transportation and store.

(7) The preparing method comprises easy procedures, costs low, and iseasy for industrialized development.

(8) The clinical application is simple, and a patient customizedapplication is also available.

The additional aspects or advantages of the invention will be furthergiven in the following descriptions, and part of them will becomeobvious through the following descriptions or be understood more easilythrough the practice.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages or the additionalaspects and advantages of the invention will be more obvious and easierfor being understood through the following descriptions in conjunctionwith the drawings and embodiments, wherein:

FIG. 1 is an illustration of electrospinning process to prepare theartificial dura mater according to the invention.

FIG. 2 illustrates of the process for preparing the artificial duramater according to the invention that combines electrospinning withbio-printing.

FIG. 3 is an illustration of the artificial dura mater according to theinvention comprising the hydrophobic electrospun layer(s).

FIG. 4 is an illustration of the artificial dura mater according to theinvention comprising the hydrophobic electrospun layer and thehydrophilic electrospun layer.

FIG. 5 is an illustration of the artificial dura mater according to theinvention comprising hydrophobic layer, hydrophilic layer and transitionlayer.

FIG. 6A is an illustration of the blended artificial dura materaccording to the invention comprising hydrophobic electrospun layer andhydrophilic electrospun layer

FIG. 6B is a magnified illustration of the Zone I of FIG. 6A.

FIG. 7A is an illustration of the blended artificial dura materaccording to the invention comprising hydrophobic electrospun layer,hydrophilic electrospun layer and transition layer.

FIG. 7B is an enlarged illustration of Zone II of FIG. 7A.

FIG. 8A is an illustration of the artificial dura mater according to theinvention obtained combining with bio-printing technology.

FIG. 8B is an enlarged illustration of Zone III in FIG. 8A.

In the figures, the numbers represent in this way.

-   -   1. Electrospinning sprayer;    -   2. Spinning fibers;    -   3. High voltage power source;    -   4. Receiving device;    -   5. Bio-printer head;    -   6. Vessel;    -   7. Hydrophobic spinning thread;    -   8. Medicine;    -   9. Hydrophilic spinning fibers;    -   10. Cytokine or medicine;    -   11. Spinning fibers of transition layer;    -   A. Hydrophobic electrospun layer;    -   B. Hydrophilic electrospun layer;    -   C. Transition layer.

DETAILED DESCRIPTION

Now, examples of the invention will be explained in detail. Theillustration of examples will be shown in the accompanying drawings, inwhich the same number represents the same or similar elements. Theexamples described with reference to the drawings are onlyillustrations, which intend for explaining the invention and do notlimit the invention.

With reference to FIGS. 1 to 8, the artificial dura mater andmanufacturing methods therefor are described in details below.

An illustration of electrospinning process to prepare the artificialdura mater is shown in FIG. 1, the electrospinning sprayer 1 containsthe polymer solution, the high voltage power source 3 has its highvoltage end connected to the sprayer 1. The receiving device 4 is incylinder shape, and can be moved leftwards and/or rightwards, along withthe axis of cylinder or the long shaft direction of cylinder. Themovement of receiving device 4 can be set with computer program, so thatthe formed electrospun layer will have equal thickness. In practice, thereceiving device can be set as a level-off surface, and through themovement between left and right or fore-and-aft, an even reception canbe realized. The receiving device 4 is connected with the low voltageend of high voltage power source 3, so that there is a large voltagedifference between the sprayer 1 and receiving device 4.

Before the electrospinning starts, the proper polymer solution forelectrospinning should be prepared.

It is an option to choose the solution of hydrophobic polymer forelectrospinning, and such solution is prepared by dissolving ahydrophobic polymer into a solvent. Wherein the hydrophobic polymerincludes but not limited to the hydrophobic aliphatic polyester(covering polylactic acid, polyglycolide, polycaprolactone, andpolyhydroxybutyrate), polyetherester (such as polydioxanone),polyorthoester, polyurethane, polyanhydride (such as poly(sebacicacid-hexadecanedioi acid anhydride)), polyphosphazene, polyamino acidand mixtures thereof.

Depending on the design, if the hydrophilic electrospun layer is neededafter the hydrophobic electrospun layer is finished, the solution ofhydrophilic polymer for electrospinning should be prepared. Wherein thehydrophilic polymer includes but not limited to the chondroitinesulfate, heparin, agar, glucan, algin, modified cellulose, alginate,starch, cellulose, gelatin, fibrinogen, silk protein, elastin-mimicrypeptide polymer, collagen, chitosan, modified chitosan, hydrophilicpolyurethane, polyethylene glycol, polymethylmethacrylate, PHBV, PHBHHx,polyvinyl alcohol and polylactide. Based on the actual needs, multiplesprayers 1 may be set, in which the hydrophobic polymer solution and thehydrophilic polymer solution are placed respectively. Or, replace thesolution in the sprayer 1 after the hydrophobic layer is made.

The solvent of the solution for electrospinning can be water or avolatile organic solvent which includes but not limited to methanoicacid, acetic acid, ethyl alcohol, acetone, dimethyl formamide, dimethylacetamide, tetrahydrofuran, dimethyl sulfoxide, hexafluoro isopropylalcohol, trifluoroethyl alcohol, dichloromethane, trichloromethane,methyl alcohol, ethyl alcohol, chloroform, dioxane, trifluoroethane, andtrifluoroacetic acid.

Once the solution for electrospinning is ready, the parameters should beset. After that, the power is on, and the electrospinning device isactivated. As the spinning fibers 2 is spun from the sprayer 1, thereceiving device 4 will move in prescribed procedures, so as to form theuniform electrospun membrane structure.

The parameters of the process to form the hydrophobic electrospun layerare set as follows: a micro-injection pump operated at a velocity of0.1-5.0 milliliters/hour and a high voltage generator operated at avoltage of 5-40 kilovolts and with a receiving distance of 5.0-30.0centimeters. The hydrophobic electrospun layer comprises fibers with adiameter, which can be controlled, ranging from 50 to 1000 μM, and poresof a size of less than 3 μM.

The parameters of the process to form the hydrophilic electrospun layerare set as follows: a micro-injection pump operated at a velocity of0.1-20.0 milliliters/hour and a high voltage generator operated at avoltage of 10-45 Kilovolts and with a receiving distance of 5.0-30.0centimeters. The hydrophilic electrospun layer comprises fibers with adiameter, which can be controlled, ranging from 5 to 200 μM and pores ofa size of 20-200 μM.

In practice, the above procedures can be repeated, so as to formmultiple hydrophobic layers and/or multiple hydrophilic layers, as shownin FIG. 3 and FIG. 4.

FIG. 3 gives an artificial dura mater with three hydrophobic layers thestrength of which is similar to the human's dura mater. As the layersare formed by hydrophobic materials, they are not good for the migrationand attachment of cells. Together with the fact that the materials aresafe, poison-free and absorbable for human body, they reach the goal ofanti-adhesion.

FIG. 4 gives an artificial dura mater consisting of two hydrophobiclayers (A) and three hydrophilic layers (B). When the dura mater istransplanted into brain, the anti-adhesive hydrophobic layers (A) areset near to the brain surface, while the hydrophilic layers (B) are setfar from the brain surface, providing a fine nanofibrous scaffold forthe adhesion, migration, proliferation and differentiation of cells. Asthe hydrophilic layers are prepared with hydrophilic materials thatoffers good bio-compatibility and have larger pore size, which is goodfor the migration of stem cells and fibroblast, consequently, which isfavorable for the growth of autologous dura mater.

Once the electrospun layers are made, they will be dried in an oven orin natural manner, dependent on varied solution component. When thesolvent of an electrospinning solution is a volatile organic solvent,such as hexafluoro isopropyl alcohol, the procedure for dryness can beomitted since the solvent has completely volatilized while the spinningfibers 2 are spun to the receiving device 4 along with a voltagedifference.

As the hydrophobic layer is very different from the hydrophilic layer inthe terms of hydrophilicity, the structural stability is not easy to bekept in the application. To solve such problem and increase thehydrophilicity between the two, a transition layer can be applied. Thetransition layer has a hydrophilicity that gradually increases from theside proximate to the hydrophobic electrospun layer to the sideproximate to the hydrophilic electrospun layer. In practice, theelectrospinning solution of the transition layer can comprise one ormore polymers and corresponding solvent which are determined based onthe requirement of hydrophilicity. Then, the solution is placed into thesprayer, by the method the as above, to prepare the transition layerbefore the hydrophilic layer. The parameters of the process to form thetransition layer are set as follows: a micro-injection pump operated ata velocity of 0.1˜5.0 milliliters/hour and a high voltage generatoroperated at a voltage of 5-40 kilovolts and with a receiving distance of5.0-30.0 centimeters.

The artificial dura mater with transition layer is illustrated in FIG.5. In the figure, the hydrophobic layer is of two layers, and thehydrophilic layer is of three layers. Between the hydrophobic layers (A)and the hydrophilic layers (B), there is a transition layer of twolayers (C), the one, proximate to the hydrophobic layers (A) has aweaker hydrophilicity than the other proximate to the hydrophiliclayers.

Furthermore, to realize the addition of cytokine and/or medicine intothe artificial dura mater, a blending electrospinning can be adopted.Specifically, a cytokine and/or a medicine can be blended into any oneor more layers of the hydrophobic, hydrophilic and transition layers. Acytokine and/or medicine should be put into a corresponding solution.After that, the electrospinning is made and the cytokine and/or medicinewill be blended into the spinning fibers 2 as the spinning fibers areformed. The membrane structure will be formed on the receiving device 4.Likewise, this procedure can be repeated. The cytokine and/or medicineadded each time can be the same or different. The acquired artificialdura mater is shown in FIG. 6. In FIG. 6A, a hydrophobic layer is of atwo-layer structure and a hydrophilic layer is of a three-layerstructure. In FIG. 6B, a hydrophobic spinning fiber 7 contains amedicine 8, and a hydrophilic spinning fiber 9 is blended with acytokine 10.

FIG. 7A has two transition layers C based on FIG. 6. One of themproximate to the hydrophobic layer A, has a weaker hydrophilicity thanthat proximate to the hydrophilic layer B. From FIG. 7B, the hydrophobicspinning fiber 7 is blended with a medicine 8, and the hydrophilicspinning fiber 9 is blended with a cytokine 10, the spinning fibers oftwo transition layers 11 is respectively blended with medicine 8 andcytokine 9.

Besides, the invention provides a method that combines electrospinningand bio-printing to prepare artificial dura mater. The bio-printing isan emerging technology, and just have been developed and applied inbiomedical fields in recent years. This technology utilizes a specialcell solution or a biological active solution as the “bio-ink” and asdesigned, prints precisely on the spot of a specific substrate (termedas “bio-paper”) that can be degraded in the human body. After printing,the bio-papers will be stacked in certain sequence. As the printingtechnology is used, the bio-ink consisting of cells and/or cytokine canbe precisely placed to the designed areas. The bio-papers, if stacked inparticular way, will form the three dimensional structure.

The specific implementation is given in FIG. 2. Based on the devicegiven in FIG. 1, the bio-printer head 5 is set, and such head can beobtained by modifying the present commercial inkjet printer as themethod, for example, disclosed in U.S. Pat. No. 7,051,654. The headcontains a cytokine and/or a medicine. The printing manner and printingposition can be set through the computer program in advance. Thespecific printing procedures can base on the current technologies.

In accordance with one embodiment of the invention, the cytokine and/ormedicine can be enclosed in hydrogel. The hydrogel solution can beaqueous solution of polysaccharide polymer, polypeptide polymer orsynthetic hydrophilic polymer. The polysaccharide polymer includes butnot limited to the starch, cellulose, alginate, hyaluronic acid andchitosan. The polypeptide polymer includes but not limited to thecollagen, poly-L-lysine and PLGA. The synthetic, hydrophilic polymerincludes but not limited to polyacrylic acid, polymethacrylic acid,polyacrylamide and N-isopropyl acrylamide. The hydrogel is liquid innormal circumstances and becomes gel at certain temperature or underspecific conditions. With which, the hydrogel possesses good adhesion,which can make a cytokine and/or a medicine evenly or definitelydistribute on the electrostatic spinning layer.

The procedures of bio-printing with hydrogel are as follows: 1) Put thecytokine and/or medicine, prepared and blended with liquid hydrogel,into the bio-printer head 5. 2) after the electrospun layer is formed,Print on the electrospun layer with jet printer as per the presetprogram; based on the choice of hydrogel, apply proper conditions tomake the hydrogel quickly become gel/jelly that, by offering goodadhesion, adhere the cytokine and/or medicine enclosed to theelectrospun layers. 3) Set even and uniform distribution of bio-ink andform the artificial dura mater as shown in FIG. 8A, in which, each ofhydrophobic layers is printed with a layer of medicine and each ofhydrophilic layers is printed with a layer of cytokine. As shown inenlarged FIG. 8B, the film prepared with bio-printing is different fromthat prepared with blending. A cytokine and/or a medicine are applied onthe surface of layers that is formed by the hydrophobic spinning fiber 7and/or the hydrophilic spinning fiber 9. When such dura mater istransplanted into human body, the cytokine and/or medicine can be evenlyreleased. 4) If necessary, set the concentrated distribution on certainpoints and make the cytokine and/or the medicine printed into particularareas. When such dura mater is transplanted to the human body, thecytokine and/or the medicine can be mostly released to the wantedspecific areas.

The solidification of some hydrogels needs a cross-linking agent toassist. In this case, a bio-printing in the utilization of hydrogel hasthe following procedures: 1) Put a certain amount of cross-linking agentinto the vessel 6. Once the electrospinning starts, the receiving device4, moving along with the axis or between left and right, will contactthe agent, and the formed electrospun layers will adhere with somecross-linking agent 2) Make the bio-printing as given above. When theliquid hydrogel inside the head 5 contacts the cross-linking agent onthe electrospun layers, the hydrogel quickly turns to gel/jelly andenables the cytokine and/or medicine enclosed to adhere to theelectrospun layers. The selection of cross-linking agent depends on thekind of hydrogel. For instance, when the hydrogel is sodium alginate,the cross-linking agent is calcium chloride; when the hydrogel isfibrinogen, the agent is thrombin.

According to the one embodiment of the invention, the solution ofhydrophobic polymer can be hydrophobic poly(L-lactide) (PLLA) andε-caprolactone dissolved to hexafluoro isopropyl alcohol ordichloromethane, the ratio of the two polymers can be 50:50, 30:70 or70:30. As a copolymer, the number-average molecular weight is150,000-500,000. When blending is needed, the hydrophobic solution canbe added with 0.01-3% antibiotic solution and/or with 0.001-3% medicinefor hemostasis and anti-adhesion. Together with the solution ofpoly(L-lactide) (PLLA) and ε-caprolactone, the final solution will beobtained.

According to another embodiment of the invention, the solution ofhydrophilic polymer can choose hydrophilic polyurethane plus naturalgelatin, chondroitine sulfate or polyethylene glycol as solvent(s). Themass ratio is 20-80:80-20. The spinning solution accounts for 3-15% oftotal weight. In blending, the solution of hydrophilic polymer can beadded with a solution of basic fibroblast factor, and make theconcentration of cytokine of 0.001-0.5%.

A medicine added in blending or bio-printing can choose, in accordancewith the actual situation, antibiotic or drug for hemostasis oranti-adhesion. In the transplantation of dura mater due to tumorexcision, the drugs for chemotherapy of brain tumor can be added.

An antibiotic includes but not limited to the cephalosporin, ampicillin,spiramycin, sulfonamides and quinolones. The first choice is ceftriaxonesodium. As meninges surgeries often need to open the skull and atpresent, the intracranial infection is often bacterial mainly comprisingstaphylococcus aureus, streptococcus, pneumococcus, escherichia coli,salmonella and pseudomonas aeruginosa. The commonest virus isstaphylococcus aureus. According to the clinical reports, theceftriaxone sodium offers better therapy effect.

The anti-tumor medicine includes but not limited to nimustine,semustine, liposome doxorubicin, dactinomycin D and vincristine. Thevincristine is first choice.

A medicine for hemostasis or anti-adhesion can speed up the healing ofwound and prevent the occurrence of adhesion. Such medicine includes butnot limited to the hemostasis factor (which makes the material own thefunction of hemostasis), the inhibitor of collagen synthase (such astranilast and pemirolast, which inhibit the revival of collagen), theanti-coagulation drug (such as dicoumarolum, ehparin sodium andhirudin), the anti-inflammatory drug (such as promethazine,dexamethasone, hydrocortisonum, prednisolone, ibuprofen andoxyphenbutazone), the calcium channel blocker (such as diltiazemhydrochloric, nifedipine and verapamil hydrochloride), the cell growthinhibitor (such as fluorouracil), the hydrolase (such as hyaluronidase,streptokinase, urokinase, pepsin and tPA) and the oxidation reductant(such as methylene blue).

According to an embodiment of the invention, in blending, theelectrospinning solution is added with a cytokine and/or a medicine inthis formula: the basic fibroblast growth factor accounting for0.001-0.05% of electrospinning solution weight, the ampicillinaccounting for 3% of electrospinning solution weight, the hemostasisfactor accounting for 0.001-0.05% of electrospinning solution weight. Ina meninges restoration operation due to brain tumor, the nimustineaccounting for 0.01-5% of the weight can be added.

The obtained artificial dura mater should be rinsed, sterilized,packaged and stored.

Example 1

Poly(L-lactide) (PLLA) and ε-caprolactone, in the mass ratio of 50:50and with number-average molecular weight of 260,000, are dissolved intoa solvent of hexafluoro isopropyl alcohol to form a hydrophobicelectrospinning solution. Put the solution into the sprayer ofelectrospinning. The micro-injection pump is operated at a velocity of 5milliliter/hour; the high voltage generator is operated at a voltage of30 kilovolts and a receiving distance of 20 centimeters. The fibers arereceived to form a membrane structure and have a diameter in the averageof 300 nm. After the receiving process is completed, the spinning deviceis closed.

The artificial dura mater acquired is rinsed for five times with ethylalcohol and distilled water. The dura mater, then, is packaged in vacuumafter freeze dry. After sterilization with 25 kGy cobalt-60, the duramater is stored at minus 20° C.

Example 2

The preparing method is the same as which of the example 1.

The hydrophilic solution for electrospinning choose poly ethanedioicacid and chondroitine sulfate, in the mass ratio of 70:30 and the massfraction of spinning liquid is 9%.

The electrospinning device is activated, and a hydrophilic electrospunlayer is formed on the hydrophobic layer already formed in Embodiment 1.The receiving distance is 11 centimeters, the voltage is 20 kilovolts,and the acquired hydrophilic layer comprises fibers in the average witha diameter of 10 μm.

The rinse and store are the same as those in Embodiment 1.

Example 3

The hydrophobic electrospun layer is prepared in the same manner withthat in Embodiment 1.

The transition layer adopts a polymer solution of polyurethane andhyaluronic acid in the mass ratio of 70:30 and has a mass fraction of10%. The spinning is activated with a receiving distance of 11centimeters and a voltage of 20 kilovolts. The fibers have a diameter ataverage of 5 μm. On the hydrophobic layer, the transition layer is made.

Then, on the transition layer, the hydrophilic layer is spun, in thesame manner as that in Example 4. After that, the spinning is stopped.

The rinse and store are the same as them in Example 1.

Example 4

(1) prepare the hydrophobic electrospun layer: Choose the hydrophobicpolycaprolactone and a mixed solvent of chloroform or methyl alcohol ina ratio of 1:1. Add with ceftriaxone sodium, at a concentration of 1%.Get the uniform solution.

Add the above solution into the sprayer for electrostatic spinning, andperform the electrospinning with a micro-injection pump operated at avelocity of 0.8 milliliters/hour and a high voltage generator operatedat a voltage of 12 kilovolts and with a receiving distance of 15centimeters. Obtain the fibers in membrane structure. The fibers of thehydrophobic electrospun layer have a diameter of 600 nanometers.

Close the spinning device.

(2) prepare the hydrophilic electrospun layer: Adopt the hydrophilicsilk protein and natural gelatin in the ratio of 20-80:80-20 and havethe mass fraction of spinning liquid at 9%.

Prepare the solution of basic fibroblast factor. Mix the solution withthe above mentioned electrospinning solution evenly, and have the finalconcentration of cytokine to be 0.001%, the receiving distance to be 10cm, and the voltage to be 20 KV. Start the electrospinning, and form thehydrophilic layer on the hydrophobic layer formed already. The averagediameter of fibers from the hydrophilic layer is in the level of micron.

The rinse and store are the same as them in Example 1.

Example 5

(1) prepare the hydrophobic electrospun layer: Select the hydrophobicpolycaprolactone and a mixed solvent of chloroform and methyl alcohol inratio of 1:1. Blend with vincristine at the concentration of 100 ng/ml,and get the uniform solution.

Add the above solution into the sprayer for electrospinning, adjust thevelocity of micro-injection pump to be 0.8 ml per hour, the voltage ofhigh voltage generator to be 12 KV, and the receiving distance ofreceiving device to be 15 cm, and obtain the fibers in membranestructure. The fibers of the hydrophobic electrospun layer have adiameter of 600 nanometers.

Close the spinning device.

(2) prepare the transition layer: Choose polyurethane and hyaluronicacid in the mass ratio of 70:30. Have the mass fraction of spinningsolution at 10%. Blend with ampicillin at concentration of 3%. Theuniform solution is form.

Start the spinning. Spin the transition layer on the hydrophobic layeralready formed.

The receiving distance is 11 centimeters, the voltage is 20 kilovolts,and the average diameter of fibers is 5 μm.

Close the spinning device.

(3) prepare the hydrophilic electrospun layer: Adopt the hydrophilicsilk protein and natural gelatin in the ratio of 20-80:80-20 and havethe mass fraction of spinning solution at 9%. Blend with ampicillinsolution at a final concentration of 3%.

Activate the spinning device, adjust the receiving distance to be 10 cm,and spin the hydrophilic layer on the transition layer already formed.The average diameter of fibers should be in the level of micron.

The rinse and store are the same as them in Example 1.

Example 6

(1) prepare the hydrophobic electrospun layer with bio-printing:

The hydrophobic solution selects the hydrophobic poly(L-lactide) (PLLA)and ε-caprolactone, in a mass ratio of 50:50, as copolymer with anumber-average molecular weight of 260000, dissolved into the hexafluoroisopropyl alcohol.

The cross-linking agent selects 0.1M calcium chloride solution.

The hydrogel containing a cytokine adopts an alginate solution ofhemostasis factor. The hemostasis factor has a concentration of 10 ppmin mass in the cytokine alginate solution.

The solution with 0.1 M calcium chloride should be placed into the cellpetri dish with a diameter of 150 mm. The co-receiver share by thespinning device and bio-printing is placed into the petri dish. Informing of an electrospun layer, the receiving device should contactwith the solution in petri dish. The printer head is fixed below theelectrospinning needle which is inside the spinning device box, whichoffers a function of fixed-point print on specific area with hemostasisfactor. The prepared cytokine alginate solution is put into thecartridge of jet printer. In this embodiment, the cartridge is HP51626A.

The hydrophobic electrospinning solution is placed into the sprayer forspinning After that, adjust the velocity of micro-injection pump to be 5milliliters/hour, the voltage of high voltage generator to be 30kilovolts, and the receiving distance of receiving device to be 20centimeters. The fiber is received in membrane structure. The spinninglasts for 20 minutes before the spinning device is closed.

The hydrogel solution containing the cytokine is printed on thenano-bionic scaffold with jet printer. Once the hydrogel is solidified,the hydrophobic electrospun layer with cytokine by bio-printing isacquired.

(2) prepare the hydrophilic electrospun layer with bio-printing:

An electrospinning solution, a hydrogel solution with medicine and thecross-linking agent solution are prepared.

Polyglycol and chondroitine sulfate, as hydrophilic materials, in themass ratio of 70:30 are chosen. The spinning solution has the massfraction of 9%. The cross-linking agent solution is the 0.1M calciumchloride solution.

The hydrogel solution containing a cytokine adopts the alginate solutionwith basic fibroblast factor. The basic alginate solution has the basicfibroblast factor at the concentration of 100 ppm.

The parameters are set as follows: Adjust the velocity ofmicro-injection pump to be 0.8 milliliters/hour, the voltage of highvoltage generator to be 20 kilovolts and the receiving distance ofreceiving device to be 11 centimeters. Other preparing procedures arethe same as those in first step above. After the hydrophilic fiber isreceived in membrane structure, by bio-printing, the hydrogel solutioncontaining the basic fibroblast factor is printed to the nano-bionicscaffold. Once the hydrogel is solidified, the hydrophilic electrospunlayer with cytokine by bio-printing is acquired.

The rinse and store are the same as those in Example 1.

Example 7

(1) The preparation of hydrophobic electrospun layer with bio-printingis the same as described in Example 6.

(2) prepare the transition layer by bio-printing

The electrospinning liquid solution of transition layer is comprises thepolyurethane and the hyaluronic acid in the mass ratio of 70:30. Themass fraction of the spinning solution is 10%. The blended ampicillin isin the concentration of 3%.

The cross-linking agent solution is the 0.1M calcium chloride solution.

The hydrogel solution with cytokine is an alginate solution ofhemostasis factor, and the mass percentage of the hemostasis factor is10 ppm.

The parameters are adjusted in this way as follows: The velocity ofmicro-injection pump is 4 milliliters/hour, the voltage of high voltagegenerator is 20 kilovolts, and the receiving distance is 11 centimeters.The other procedures are the same as the above procedures. The fiber isreceived in membrane structure. The hydrogel solution containing thecytokine will be printed on the transition layer. After the hydrogel issolidified, the hydrophobic electrospun layer with cytokine bybio-printing is obtained.

(3) The preparation of hydrophilic electrospun layer with bio-printingis the same as described in Example 6.

The rinse and store are the same as those described in Example 1.

Experimental Example 1

The dura mater obtained from Example 1 is applied to dog for the animalexperiment, while the control is a commercialized, clinical applicableheterogenic meninges repairing product. Three healthy dogs are chosen,either male or female, in the weight between 10 and 15 kg, and underobservation for two to three months. The dogs chosen in the experimentare under general anesthesia. Their skulls are opened at both left andright sides. Their dura maters are surgically removed, and the defectsof dura maters and injuries of brain tissue appear. Then, the duramaters obtained from the Embodiment 1 and the control, respectively, areimplanted for repairing the dural defects at the left and right sides ofthe dog's brain. After operation, the dogs are feed normally as usualand observed periodically. The specimen is taken from the implanted areaat the end of each observation period. In the end of the observationperiod, the skulls of the dogs are exposed by the way described aboveafter general anesthesia. Expose and separate the outer surface of therepairing material. To retrieve the samples, these dogs are sacrificedby administration of an intravenous injection of air. The skulls areopened surgically. The implants and surrounding tissue are taken out.The retrieved samples are inspected carefully with details including theappearance, characters, its relation to the surroundings, cyst,callosity, as well as the adhesion between its inner surface and braintissue. The specimen is stored in a bottle and treated and fixed withFormalin solution. Label the bottle. One week later, take out a localtissue; embed with paraffin section and histological stain with HE.

Several days after implantation, the three dogs recover well, andincisions are healed well with no obvious secretion. The dogs eat anddrink normally as well as their outdoor activities are normal withoutany obvious movement malfunction. Three months post-implantation, thethree dogs are sacrifice by an intravenous injection of air. Aftersacrifice, taking the surgical site as center, the specimen is cut fromone centimeter outer the operation site, and it includes the repairmaterial, the surrounding dura mater and part of brain tissue. After thespecimen is taken, separate the skull and meninges in order. It is foundthat the meninges, where dura mater transplanted, are formed completely;the transplanted material has been replaced by fibrous tissue, and hasconnected tightly with the native meninges without border. The internalsurface of the newly-formed meninges at transplantation site is freefrom adhesion with the brain tissue, and the surface of correspondingbrain tissue is smooth and free from adhesion to the implant. While, inthe implanted site of the control samples, the transplanted material hasnot yet degraded and exist a few of adhesion at the transplantation siteto the internal surface of meninges.

Experimental Example 2

The dura mater prepared in the Example 2 is now applied for the dogexperiment. Five dogs are in weight of 15-20 kg, 1.5-2-year-old, andeither male or female. They are in general anesthesia through theintramuscular injection of ketamine. After anesthesia and shave, theanimals are placed on the operation table in lateroabdominal position.The disinfection is made with 2% iodine and 75% alcohol. The animals'heads are opened surgically in lengthwise direction. Stripper is used toseparate the periosteum of the skull, and the two top skull plateaus areexposed. The high-speed driller is used to open the skull. The two skullwindows at vertex are formed, and two rectangular dura maters in size of3 cm×3 cm at the top of head are cut off with the scissors. Finally thetop dura mater defects are made for the following implantation withartificial dural mater and control. On the exposed brain surface, theelectrocoagulation is applied to make 6 injury points in the size of 1mm×1 mm. Then, the artificial dura mater manufactured in Embodiment 2 istrimmed to the same shape and size as those of the dural defect andloaded into the defect. The hydrophobic layer is toward the brainsurface, and the sutures is made with the 4/0 thread in the interval of4 mm, to repair the dural defect of dogs. The round needle and 4/0thread are used for suture of muscle. The commercialized, clinicallyapplicable, animal material based dural repair product is applied as acontrol. After operation, the dogs are feed normally as usual andobserved periodically. The animals recover well, and incisions arehealed well. No obvious leakage of cerebrospinal fluid or occurrence ofepilepsy is found. The dogs eat and drink normally as well as theiroutdoor activities are normal without any obvious movement malfunction,and they survive to the expected longevity.

Eighteen months post-implantation, the dogs are sacrificed and thespecimen is taken at the surgical site, and it includes the artificialdura mater, the surrounding dura mater, and part of the surroundingbrain tissue. After carefully observe the specimen, we can see that theconnection between the artificial dura mater and native dura mater istight and smooth, without clear boundary, healed well, besides, and withthread seen only. The native dura mater does not show any hyperaemia,hemorrhage or other rejection reaction. While, the results of thecontrol show that the implant material is not yet degraded, and at theimplanted site, the inner surface of meninges adheres to brain tissue tosome degree.

Experimental Example 3

The artificial dura mater obtained from Example 3 is now under theexperiment with New Zealand rabbit.

The animals under experiment are opened in the skulls, and the defect ofdura mater and injury of brain tissue are made surgically. Then, theartificial dura mater is used to repair the defect. After the operation,the rabbits are feed normally as usual and observed periodically. Theseanimals recover well. Eighteen months post-implantation, the rabbits aresacrificed and the specimen is taken at the surgical site. The specimenincludes the artificial dura mater, surrounding dura mater and part ofthe surrounding brain tissue. When observe the specimen carefully, it isseen that the epithelial cells cover the inner surface of the duramater; under the epithelium, fibrous tissue is formed, fibroblastprogenitor cells proliferate, and collagen fibers are increased. All ofthese result in the formation of new vascularized tissues, in-growth ofnative dura mater, degradation of the implant material, deduction oftotal mass of the implant, and generation of rich capillary networks. Inthe interface of old and new tissues, no neutrophil, lymphocyte or othercell reaction for inflammation is found, and no cyst wall is formed. Thearachnoid mater and brain tissue are normal.

Experimental Example 4

The dura mater obtained from Example 4 is now applied to the dogexperiment, and the method is the same as Experimental example 2.

Fifteen months post-implantation, the dogs are sacrificed and thespecimen is taken at the surgical site. The specimen includes theartificial dura mater, surrounding dura mater, and part of thesurrounding brain tissue. After carefully observe the specimen, it isseen that the connection between the artificial dura mater and nativedura mater is tight and smooth, without clear boundary, completed cured,and with thread seen only. The native dura mater does not showhyperaemia, hemorrhage or other rejection reaction.

Experimental Example 5

The dura mater obtained from Example 5 is now applied to the New Zealandrabbit experiment, and the control is the commercialized, clinicallyapplicable, animal materials based dura mater product as the repairmaterial.

The method is the same as Experimental example 3. Fifteen monthspost-implantation, the rabbits are sacrificed and the specimen is takenat the surgical site. When observe the specimen carefully, it is seenthat the epithelial cells cover the inner surface of the dura mater;under the epithelium, fibrous tissue is formed, fibroblast progenitorcells proliferate, and collagen fibers are produced. All of these resultin the formation of new vascularized tissues, in-growth of native duramater, degradation of the implant material, deduction of total mass ofthe implant, and generation of rich capillary networks. In the interfaceof old and new tissues, no neutrophil, lymphocyte or other cell reactionfor inflammation is found, and no cyst wall is formed. The arachnoidmater and brain tissue are normal. While, the results with the controlshow that the implant material is not yet degraded, and at the implantedsite, the inner surface of meninges adheres to brain tissue to somedegree.

Experimental Example 6

The dura mater obtained from Example 6 is now applied to the dogexperiment, and the method is the same as Experimental example 2.

Twelve months post-implantation, the dogs are sacrificed and thespecimen is taken at the surgical site. The specimen includes theartificial dura mater, surrounding dura mater, and part of thesurrounding brain tissue. When observe the specimen carefully, it isseen that the connection between the artificial dura mater and nativedura mater is tight and smooth, without clear boundary, completed cured,and with thread seen only. The native dura mater does not showhyperaemia, hemorrhage or other rejection reaction.

Experimental Example 7

The dura mater obtained from Example 7 is now applied to the New Zealandrabbit experiment.

The method is the same as Experimental example 3. Twelve monthspost-implantation, the rabbits are sacrificed and the specimen is takenat the surgical site. When observe the specimen carefully, it is seenthat the epithelial cells cover the inner surface of the dura mater;under the epithelium fibrous tissue is formed, fibroblast progenitorcells proliferate, and collagen fibers are increase. All of these resultin the formation of new vascularized tissues, in-growth of native duramater, degradation of the implant material, deduction of total mass ofthe implant, and generation of rich capillary networks. In the interfaceof old and new tissues, no neutrophil, lymphocyte or other cell reactionof inflammation is found, and no cyst wall is formed. The arachnoidmater and brain tissue are normal.

Notwithstanding the embodiments have been given and described, theperson skilled in the field can, without deviation from the principleand theory, can make the variation, modification, replacement and changeof these embodiments, as given in the invention scope and right claims.

1.-35. (canceled)
 36. A method of treating a subject having a defectivedura mater, comprising implanting an artificial dura mater proximate tosaid defective dura mater in the brain of said subject, wherein saidartificial dura mater consists essentially of synthetic materials andcomprises at least a hydrophobic and biodegradable electrospun layercomprising 30% or more polylactic acid (PLA), and wherein saidhydrophobic electrospun layer comprises fibers with a diameter of50-1000 nM and pores with a size of less than 3 μM.
 37. The methodaccording to claim 36, wherein said artificial dura mater furthercomprises at least a hydrophilic electrospun layer disposed on saidhydrophobic electrospun layer.
 38. The method according to claim 37,wherein said hydrophilic electrospun layer is prepared, by a methodcomprising electrospinning, with one or more hydrophilic polymersselected from the group consisting of chondroitine sulfate, heparin,agar, glucan, algin, modified cellulose, alginate, starch, cellulose,gelatin, fibrinogen, silk protein, elastin-mimicry peptide polymer,collagen, chitosan, modified chitosan, hydrophilic polyurethane,polyethylene glycol, polymethylmethacrylate,poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV),poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), polyvinylalcohol, polylactide and mixtures thereof.
 39. The method according toclaim 37, wherein said artificial dura mater further comprises atransition layer disposed between the hydrophobic and hydrophilicelectrospun layers, wherein said transition layer has hydrophilicitythat gradually increases from the side proximate to said hydrophobicelectrospun layer e side proximate to said hydrophilic electrospunlayer.
 40. The method according to claim 36, wherein said artificialdura mater further comprises a cytokine and/or a medicine that isadhered to said hydrophobic electrospun layer and/or said hydrophilicelectrospun layer.
 41. The method according to claim 40, wherein saidcytokine is selected from the group consisting of interleukin, colonystimulating factor, tumor necrosis factor, platelet derived growthfactor, basic fibroblast growth factor and combinations thereof.
 42. Themethod according to claim 40, wherein said medicine is one or moreselected from the group consisting of an antibiotic, a hemostat, ananti-adhesion agent and an anti-tumor drug.
 43. The method according toclaim 40, wherein said cytokine and/or medicine is enclosed in ahydrogel.
 44. The method according to claim 43, wherein said hydrogelcomprises one or more selected from the group consisting ofpolysaccharide polymer, polypeptide polymer and synthetic hydrophilichigh molecular polymer.
 45. A method of treating a subject having adefective dura mater, comprising implanting an artificial dura materproximate to said defective dura mater in the brain of said subject,wherein said artificial dura mater consists essentially of syntheticmaterials and comprises at least a hydrophobic and biodegradableelectrospun layer consisting essential of polylactic acid (PLA), andwherein said hydrophobic electrospun layer comprises fibers with adiameter of 50-1000 nM and pores with a size of less than 3 μM.
 46. Themethod of claim 45, wherein the artificial dura mater is prepared by amethod comprising the steps of: (a) dissolving PLA into a solvent toobtain a electrospinning solution comprising essentially of PLA, (b)producing, by electrospinning, a film-like hydrophobic electrospun layerfrom the electrospinning solution, thereby preparing the artificial duramater.
 47. The method of claim 46, wherein at step (b) theelectrospinning is performed with a micro-injection pump operated at avelocity of 0.1-5.0 milliliters/hour and a high voltage generatoroperated at a voltage of 5-40 kilovolts and with a receiving distance of5.0-30.0 centimeters.